Partner specificity and latitudinal patterns of diversity in an algal-salamander mutualism

The mutualism between the green alga Oophila amblystomatis ((Lambert, 1905 ex Wille, 1909) and egg masses of the spotted salamander Ambystoma maculatum (Shaw) involves acquisition of algal cells from water, followed by their population growth within embryonic capsules of the egg mass. Sampling thus far has been of insufficient geographic and sequencing scale to quantify the specificity of partner choice and whether it varies latitudinally. We used PacBio and Illumina sequencing of the 18S rRNA gene to investigate diversity in egg capsule fluid harvested from five locales separated by 1° latitude. We re-analyzed 18S (V4) and chloroplast 16S (V4/V5) reads from previous work using denoising protocols, which, along with current data, produced 16.37 million reads. We detected no chlorophytes outside the Oophila clade and detected cercozoan protists and rhizophydialean fungi in some capsules. There was a mean of 10.14  3.40 amplicon sequence variants (ASVs) per sample for Oophila , ranging in prevalence from endemic to omnipresent. We observed among-locale variation in mean ASVs/sample, but not in a latitudinal pattern. However, the mean proportion of the two most prevalent ASVs varied as a function of latitude. Pairwise alignments of ASVs from the V4 region revealed an inverse relationship between sequence identity and relative abundance in read counts. We conclude that the specificity of this mutualism is high, but additional closely related ASVs, with varying relative abundance and prevalence, are detectable. This mutualism may be suited as a model with which to test theories of partner choice.